The Wnt/wingless (wg) pathway is one of a core set of evolutionarily conserved signaling pathways that regulates many aspects of metazoan development. Misregulation of the Wnt pathway can be detrimental since mutations in several components are associated with tumorigenesis of the liver, colon, breast and skin. It is therefore crucial to develop and implement new technologies in order to generate molecular tools that may be used to modulate the activity of the Wnt/wg signaling pathway. One of the most important effectors of the Wnt pathway is encoded by the transcription factor, ?-catenin (?-cat)/armadillo (arm). Since Catenin Responsive Transcription (CRT) has been implicated in the genesis of many cancers, it makes a good target for developing therapeutics that could modulate the nuclear activity of ?-cat. Recently, we employed a novel methodology of integrating a "sensitized" chemical genetic high-throughput screen (HTS) with RNA-interference (RNAi) screening technology in order to identify specific small molecule inhibitors of the Wnt pathway in Drosophila cells. Objective/hypothesis: We hypothesize that our primary chemical genetic screen will identify small molecule inhibitors that specifically target the activity of the stabilized pool of ?-cat. In this proposal, we outline experiments to investigate the molecular mechanism(s) by which the candidate small molecules impact the activity of stabilized ?-cat and also identify their protein targets. Moreover, since target identification of small molecules has been traditionally difficult, we also propose to identify the comprehensive protein "interactome" network of ?-cat using mass-spectrometry. Finally, we propose that comparative phenotypic analysis of dsRNA-mediated knockdown of the known and newly identified ?-cat-interacting proteins with that of the candidate small molecules will provide us with a novel method for target identification of the inhibitors isolated in the chemical genetic screen. Specific aims: 1) Investigate the molecular mechanisms by which the small molecules identified in the primary screen impact CRT and Wnt-responsive phenotypes in cultured cells. 2) Identify novel protein interaction partners of ?-cat/arm that may regulate the activity/stability of ?-cat. 3) Perform comparative phenotypic analysis of candidate small molecules and dsRNA-mediated knockdown of ?-cat-interacting proteins and assess whether the candidate small molecules alter the binding of the ?-cat to its known and newly identified cognate protein partners. Study design: For the chemical genetic screen, we will activate the Wnt pathway using the dsRNA-mediated knockdown of the negative regulator, Axin which results in the stabilization and activation of the cytosolic pool of ?-cat. These "activated" cells will be treated with large small molecule libraries to test if any of the individual compounds could inhibit ?-cat mediated activation of transcription (CRT) as judged by activity of the Wnt- responsive luciferase reporter gene (dTF12). The candidate small molecules will be tested for their ability to alter ?-cat's interaction with its known protein partners, such as Tcf, Bcl9/legless (lgs), pygopus (pygo), APC and Axin using FRET and co-immunoprecipitation assays. We will also perform epistasis analysis (in cells) using RNAi of known regulators of the pathway in conjunction with candidate small molecules. This will enable us to determine the site/stage at which the candidate small molecules affect the Wnt pathway. Additionally, we will identify novel protein interaction partners of ?-cat using the TAP-tag (Tandem Affinity Purification) technology with the purpose of finding additional partner proteins that might regulate the activity of the stabilized pool of??-cat. Moreover, we will test if the candidate small molecules could abrogate the interaction between ?-cat and the novel interacting proteins identified in the TAP screen. Finally, we will employ comparative phenotypic analysis to test for similarities between phenotypes obtained from small molecules or dsRNA-mediated knockdown of??-cat protein partners. We will employ cell-based and in vivo reporter assays, immuno- cytochemical and morphology-based assays in order to conduct the phenotypic analysis. Project narrative The Wnt/wingless (wg) signaling pathway is an evolutionarily conserved pathway, which is involved in the regulation of many aspects of cell biology and animal development. Misregulation of the Wnt pathway has also been implicated in a variety of human diseases including cancer of the liver, colon, breast and the skin. The primary goal of this project is to develop and implement new technologies in order to generate molecular tools that may be used to modulate the activity of the Wnt signaling pathway.